CN110291400B - Lateral flow device - Google Patents

Abstract

Lateral flow devices, methods and kits for performing lateral flow assays are provided.

Description

Lateral flow device

This application claims the benefit of U.S. provisional application 62/451,501 filed on day 27, 1 in 2017, which is incorporated herein by reference in its entirety.

Background

Methods for detecting immobilized analytes are often used in bioscience. For example, conventional blots (e.g., southern, northern, western, far Western, easter, vacuum, middle easter, easter-Western, far-easter blots, etc.) may be used to detect analytes immobilized on or in a substrate or membrane (e.g., in agarose or acrylamide). Generally, such blotting techniques involve immobilizing the analyte to be detected and contacting the analyte with a binding agent (e.g., an antibody). Blotting also typically involves multiple washing steps and/or blocking steps between immobilization and final detection. Such washing and blocking steps consume limited time and/or reagents for the practitioner and can be a source of error and non-reproducibility.

Disclosure of Invention

Lateral flow test devices and methods of using such devices are provided herein.

In one embodiment, the lateral flow device comprises a wicking pad comprising a porous material having a planar region for contacting a substrate (e.g., western blot) comprising an immobilized analyte (e.g., protein); and wherein the wicking pad has a first end, a second end, and two side edges; a support layer bonded to the first planar surface of the wicking pad; a top layer bonded to the second planar surface of the wicking pad, the top layer comprising two or more reservoirs spatially separated from each other, wherein a longitudinal axis of each reservoir is perpendicular to a side edge of the wicking pad, wherein each reservoir is in fluid communication with the first end of the wicking pad, and wherein each reservoir comprises an aperture through which solution is released from the reservoir; and a pump comprising an absorbent pad located on the second end of the wicking pad.

In some embodiments, the wicking pad further comprises two or more regions separated from each other by a hydrophobic or impermeable barrier, the barrier being parallel to the side edges of the wicking pad. In some embodiments, the edges of the wicking pad are sealed with a waterproof material or heat seal. In certain embodiments, the water repellent material is selected from the group consisting of acrylic, wax, and photopolymer.

In certain embodiments, the top layer further comprises a first opening through which the substrate contacts a planar region of the wicking pad downstream of the reservoir. In some embodiments, the top layer further comprises a second opening through which the pump contacts a second plane of the wicking pad downstream of the first opening. In some embodiments, the top layer includes an opening through which the substrate contacts the planar region and through which the pump contacts the wicking pad downstream of the planar region.

In some embodiments, the aperture is a slot. In some embodiments, the reservoirs comprise two or more sets of reservoirs spatially separated from each other and adjacent to each other on a width axis of the lateral flow device. In some embodiments, each reservoir spans the width of the wicking pad. In some embodiments, at least the first reservoir shares a wall with at least the second reservoir.

In some embodiments, the support layer and the top layer are formed of a liquid impermeable material. In certain embodiments, the liquid impermeable material is selected from the group consisting of polypropylene, polycarbonate, polystyrene, polyethylene terephthalate, and glycol modified polyethylene terephthalate.

In some embodiments, the analyte is a protein.

Methods of performing lateral flow assays are also provided. In some embodiments, the method comprises providing a lateral flow device as described above or elsewhere herein; optionally applying a running buffer to the wicking pad; applying a substrate comprising a protein (e.g., a Western blot) to the planar area for contact of the substrate; starting from the reservoir closest to the planar area, applying a different reagent solution to each reservoir for application of the substrate; and allowing the reagent solution to flow from the reservoir side to the pump such that each reagent in the reagent solution is transported in turn in the wicking pad and contacts the proteins on the substrate.

In some embodiments, different reagent solutions are applied to the reservoir sequentially or simultaneously.

In some embodiments, the step of allowing the lateral flow comprises allowing the primary antibody from the first reagent solution in the first reservoir to bind to the target protein (if present) on the substrate, and then allowing the first wash solution from the second reagent solution in the second reservoir to remove unbound primary antibody from the substrate. In some embodiments, the step of allowing the lateral flow further comprises contacting the secondary antibody or second detection reagent from the third reagent solution in the third reservoir with a primary antibody that binds to the target protein (if present) on the substrate. In some embodiments, the step of allowing the lateral flow further comprises allowing the second wash solution from the fourth reagent solution in the fourth reservoir to remove unbound secondary antibodies from the substrate.

In some embodiments, the volume of the second wash solution is at least twice the volume of the third reagent solution with the second antibody. In certain embodiments, the method further comprises optionally removing the substrate after the primary antibody binds to the target protein (if present), optionally after the secondary antibody or the second detection reagent is contacted with the primary antibody, and detecting binding of the primary antibody to the target protein (if present).

In certain embodiments, the method further comprises applying a substantially uniform pressure to the pump.

Kits for performing lateral flow are also provided. In some embodiments, the kit comprises a lateral flow device as described above or elsewhere herein. In some embodiments, the kit includes a plurality of absorbent pads that function as pumps, all of which are described herein. In some embodiments, the kit includes reagents (e.g., binding agents including labeled primary or primary and secondary antibodies, wash solutions, and/or running buffers) provided in the form of solutions to be applied to the reservoir by the end user. In certain embodiments, some or all of the reagent is dried onto the wicking pad in the portion of the wicking pad that is in fluid communication with each reservoir of the device.

In some embodiments, the kit further comprises a running buffer for performing a lateral flow, and optionally comprises a blocking agent (e.g., bovine serum albumin, skim milk powder, or casein), a surfactant (e.g., tween 20 or triton X-100), a protein aggregation modifier described herein, a macromolecular crowding agent (e.g., dextran, polyethylene glycol, and/or Ficoll), a densitometer, and/or an agent that promotes uniform flow of the agent and/or promotes reaction to molecules on the substrate and minimizes background on the substrate. The additional reagents may be provided in the kit in solid or liquid form. In some embodiments, the kit further comprises instructions for performing the methods described herein.

Drawings

FIGS. 1A and 1B are schematic cross-sectional side and top views, respectively, of a lateral flow device according to one embodiment. The device includes four reservoirs which in turn transport reagent solution to a wicking pad that is bonded to a support and the four reservoirs. The device shown has two openings in the top layer downstream of the reservoir.

Fig. 2A and 2B are schematic cross-sectional side and top views, respectively, of the device of fig. 1A and 1B, wherein the substrate and pump are in intimate contact with the wicking pad. The substrate is applied to the wicking pad through a first opening and the pump is applied to the wicking pad through a second opening downstream of the first opening.

Fig. 3a,3b and 3C are schematic cross-sectional side and top views of a lateral flow device according to one embodiment. In this embodiment, the top layer partially covers the wicking pad, i.e. only the portion of the top layer with the reservoir covers and adheres to the wicking pad. There is no top layer downstream of the reservoir, i.e. in the planar area on which the substrate and pump are applied. Fig. 3A shows a device with a pump and substrate in intimate contact with a wicking pad. Figures 3B and 3C show the device without the pump or substrate applied to the wicking pad.

FIG. 4 is a schematic top view of a lateral flow device having multiple sets of reservoirs such that multiple substrates can be analyzed simultaneously, according to one embodiment. The device shown has a plurality of substrates in intimate contact with the wicking pad downstream of the plurality of sets of reservoirs. The device also shows a belt pump in intimate contact with the wicking pad downstream of the substrate.

FIG. 5 is a schematic top view of a lateral flow device having multiple sets of reservoirs such that multiple substrates can be analyzed simultaneously, according to one embodiment. In this embodiment, the wicking pad comprises two or more regions separated from each other by a hydrophobic or impermeable barrier that is parallel to the side edges of the wicking pad. The illustrated device has a plurality of substrates in intimate contact with wicking pads downstream of the sets of reservoirs and separated from each other by barriers. The device also shows a belt pump in intimate contact with the wicking pad downstream of the substrate.

FIGS. 6A and 6B are immunoblotting results using the lateral flow device of FIGS. 1A-2B and as described in example 1.

Detailed Description

Described herein are lateral flow devices and methods of using such devices that allow for efficient lateral flow detection of analytes (e.g., proteins, nucleic acids) immobilized on a substrate (e.g., western blot membrane) or a wicking pad (e.g., diagnostic application) using specific binding agents (e.g., antibodies). The devices and methods described herein also allow for efficient lateral flow detection of analytes captured by specific binding agents immobilized on a substrate. Lateral flow devices and methods of using such devices have been discovered that sequentially and hands-free deliver different solutions (e.g., samples with one or more analytes, specific binding agents, running buffers, wash solutions) to a wicking pad in intimate contact with a substrate having the analyte or binding agent immobilized thereon. The solution is transported sequentially from at least two reservoirs bonded to a wicking pad of the lateral flow device to the wicking pad. In some embodiments, the devices described herein may be assembled in a single-use device, allowing for an economical and simple test mode.

I. Definition of the definition

The term "analyte" refers to a biological molecule, e.g., a protein, nucleic acid, polysaccharide, lipid, antigen, growth factor, hapten, etc., or a portion thereof. Analytes may be immobilized reversibly or irreversibly on a surface (e.g., a membrane or a wicking pad) and detected as described herein.

The term "immobilized" or "embedded" interchangeably refers to a molecule (e.g., an analyte or binding agent) that is reversibly or irreversibly immobilized. In some embodiments, the reversibly immobilized molecules are immobilized in a manner that allows the molecules or portions thereof (e.g., at least 25%, 50%, 60%, 75%, 80% or more of these molecules) to be removed from their immobilized locations without significant denaturation or aggregation. For example, a solution containing molecules is brought into contact with an absorbent material, thereby absorbing the solution and reversibly immobilizing the molecules, thereby reversibly immobilizing the molecules in or on the absorbent material (e.g., absorbent pad). The reversibly immobilized molecules can then be removed by wicking the solution from the absorbent material, or by wicking the solution from one region of the absorbent material to another. In some cases, the solution containing the molecules is contacted with the absorbent material to aspirate the solution, and then the absorbent material containing the solution is dried, thereby reversibly immobilizing the molecules on the absorbent material. The reversibly immobilized molecules can then be removed by contacting the absorbent material with another solution of the same or different composition, thereby dissolving the reversibly immobilized molecules, and then either wicking the solution from the absorbent material or from one region of the absorbent material to another.

The irreversibly immobilized molecules (e.g., binding agents or analytes) are immobilized such that they are not or substantially not removed from their positions under mild conditions (e.g., a pH of about 4-9, a temperature of about 4-65 ℃). Exemplary irreversibly immobilized molecules include protein analytes or binding agents that bind to nitrocellulose, polyvinylidene fluoride, nylon, or polysulfone membranes by standard blotting techniques (e.g., electroblotting). Other exemplary irreversibly immobilized molecules include protein analytes or binding agents bound to glass, plastic (e.g., microarrays, microfluidic chips, glass histological slides, or plastic microtiter plates with wells and bound protein analytes in the wells), particles, nanoparticles, or magnetic particles.

The term "binding agent" refers to a substance that specifically binds a molecule (e.g., an analyte). Although antibodies are described in much of the disclosure herein, it should be understood that other binding reagents may be used instead of antibodies, as preferred by the user. A variety of binding agents are known in the art, including antibodies, aptamers, adhesion agents (affimers), lipocalins (e.g., anti-calins), thioredoxin a, bile triene binding proteins, or proteins containing ankyrin repeats, the Z domain of staphylococcal protein a, or fibronectin type III domain. Other binding agents include, but are not limited to, biotin/streptavidin, chelators, chromatographic resins, affinity tags or functionalized beads, nanoparticles, and magnetic particles.

The term "specific binding" refers to a molecule (e.g., a binding agent such as an antibody or antibody fragment) that binds to a target with at least 2-fold higher affinity than a non-target compound, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, or 1000-fold or more.

The term "antibody" refers to a polypeptide or fragment thereof comprising a framework region from an immunoglobulin gene that specifically binds to and recognizes an antigen, e.g., a particular analyte. Typically, the "variable region" comprises the antigen binding region of an antibody (or a functional equivalent thereof) and is critical for the specificity and affinity of binding. See Paul, fundamental Immunology (basic immunology) (2003). Antibodies include, for example, chimeric, human, humanized antibodies, or single chain antibodies.

Structural units of exemplary immunoglobulins (antibodies) includeTetramer. Each tetramer comprises identical two pairs of polypeptide chains, each pair comprising one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The term light chain variable region (V _L ) And a heavy chain variable region (V _H ) These light and heavy chains are referred to respectively.

Antibodies can exist in the form of intact immunoglobulins or any of a number of well-characterized fragments that comprise specific antigen-binding activity. Such fragments may be produced by digestion with various peptidases. Pepsin digests antibodies below disulfide bonds in the hinge region, producing the dimer F (ab) of Fab' ₂ Fab itself is joined to V by disulfide bonds _H -C _H 1. F (ab') can be reduced under mild conditions ₂ To break the disulfide bond in the hinge region, thereby breaking F (ab') ₂ The dimer is converted to Fab' monomer. The Fab' monomer is essentially a Fab with a partial hinge region (see basic immunology (Fundamental Immunology), paul et al, 3 rd edition, 1993). Although various antibody fragments are defined in terms of digestion of intact antibodies, one skilled in the art will appreciate that such fragments may also be synthesized de novo using chemical methods or recombinant DNA methods. Thus, the term antibody as used herein also includes antibody fragments produced by modification of the entire antibody, or generated by head synthesis using recombinant DNA methods (e.g., single chain Fv), or those identified using phage display libraries (see, e.g., mcCafferty et al, nature 348:552-554 (1990)).

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the term "about" refers to the numbers listed as well as any value within 10% of the numbers listed. Thus, about "5" refers to any value between 4.5 and 5.5, including 4.5 and 5.5.

II. device

FIGS. 1-5 show embodiments of lateral flow devices for detecting an analyte on a substrate, for detecting an analyte bound to a binding agent on a substrate, or for detecting an analyte bound to a binding agent on a wicking pad.

Referring to fig. 1A-2B, a lateral flow device 100 includes a wicking pad 102, the wicking pad 102 having a first end 104, a second end 106, two side edges 108, and a planar region 110 for contacting a substrate 112 (e.g., a membrane) containing an immobilized analyte or protein to be detected (e.g., western blot, dot blot). The lateral flow device 100 also includes a support layer 114 and a top layer 116. The support layer 114 is bonded to the first planar surface 118 of the wicking pad 102. The top layer 116 is bonded to the second planar surface 120 of the wicking pad 102. Thus, the wicking pad 102 is sandwiched (e.g., laminated) between the support layer 114 and the top layer 116. The top layer 116 partially covers and adheres to the wicking pad 102 and prevents fluid evaporation during use of the lateral flow device 100. In some embodiments, the top layer 116 is bonded to the support layer 114 on an edge.

One end of the top layer 116 includes two or more reservoirs 122 that are spatially separated from each other. In some embodiments, the longitudinal axis of each reservoir is perpendicular to the side edges 108 of the wicking pad 102 such that each reservoir is oriented perpendicular to the direction of the side stream. A reservoir 122 (e.g., R1, R2, R3, and R4) is located at or near the first end 104 of the wicking pad 102 upstream of the planar region 110 for contacting the substrate 112. As used herein, "proximal" is defined as no more than 10%,20%,30%,40% or 50% of the length from the first end 104 of the wicking pad 102. Each reservoir 122 is in fluid communication with the first end 104 of the wicking pad 102 (i.e., liquid may flow from each reservoir 122 into the wicking pad 102 when present in the reservoir 122). The reservoir 122 sequentially supplies liquid (e.g., buffer and detection reagents) to the wicking pad 102 and into the planar region 110 to apply the substrate 112. The planar region 110 of the wicking pad 102 is located downstream of the reservoir 122 and upstream of the pump 124 (i.e., between the reservoir 122 and the pump 124).

As shown in fig. 1A-3C, the reservoir 122 includes a set of four reservoirs. In some embodiments (fig. 4 and 5), the reservoirs comprise two or more sets of reservoirs 440, 540, the reservoirs being spatially separated from each other (e.g., separated by a wall or a distance) such that multiple substrates can be analyzed at once. In some embodiments, the reservoir sets 440, 540 are adjacent to each other on the width axis of the lateral flow device 400, 500. The reservoir sets 440, 540 are arranged to extend parallel to each other in side-by-side relation. Each set of reservoirs is functionally independent of an adjacent set of reservoirs.

Referring again to fig. 1A-3C, each reservoir 122 is defined by a first wall 126 and a second wall 128 oriented perpendicular to the flow of liquid. Each reservoir is further defined by two end walls 130. In certain embodiments, the reservoirs share walls. For example, the second wall of the first reservoir R1 may be the first wall of the second reservoir R2. The reservoir 122 may be pre-molded during the manufacture of the top layer 116, or may be manufactured separately and then directly bonded to the top layer 116.

The reservoir 122 may be of any size and shape. In some embodiments, each reservoir 122 includes a length L1, a width W1, and a depth D1. In some embodiments, each reservoir is at least about 0.5,1.0,8.5, 13.5, 20cm or more in at least one dimension. In some cases, the length L1 and width W1 of each reservoir 122 are at least about 2 times, 3 times, 5 times, 10 times, 100 times, or more greater than the depth D1. In some embodiments, each reservoir is sized to match the width of the wicking pad 102 and has a length L1 that is at least about 3, 4, 5, 6, 8, 10, 13, 17, 20, 27 or more times greater than the width W1. Exemplary dimensions of each reservoir include, but are not limited to, width W1 and length L1 of about 0.5cm by 0.5cm,0.5cm by 1cm,0.5cm by 8.5cm,1cm by 3cm,3cm by 3cm,2.5cm by about 8.5cm,1cm by 10cm,3cm by 10cm,2cm by 13.5cm,3 by 13.5cm,1cm by 15cm,3cm by 15cm, or 3.5cm by 20cm, respectively. As used herein, "width W1" is based on the direction of flow and is the shortest dimension. In some embodiments, each reservoir has a width W1 of 3cm and a length L1 of 10cm. In some cases, each reagent reservoir has a width W1 of 1.+ -. 0.5,1,2 or 3cm and a length L1 of 10.+ -. 0.5cm or 15.+ -. 0.5cm. In some cases, the depth D1 of the at least one reservoir is about 0.5cm, about 1cm, about 2cm, or about 3cm. Each reservoir 122 includes an aperture 132 (e.g., a slot) through which solution is released from the reservoir and into the wicking pad 102. In embodiments where the aperture 132 is a slot, the slot may have a width W2 (e.g., short dimension) ranging from about 0.1mm to about 5mm and a length L2 (e.g., long dimension) ranging from about 0.5cm to about 20cm. In some embodiments, the width W2 is 0.1mm,0.5mm,1mm,2mm,3mm,4mm, or 5mm. In certain embodiments, the length L2 is 8.5cm,9cm,9.5cm,13.5cm,15cm, or 20cm.

The volume of each reservoir 122 may be determined by a number of factors, including, but not limited to, the size and shape of the reservoir 122 and the configuration of the lateral flow device 100. In some embodiments, each reservoir has a capacity of at least about 0.25 milliliters to about 30 milliliters. In some embodiments, the thickness of the top layer 116 defines the volume of the reservoir. In some embodiments, the top layer 116 is 3/8 inch or 1/4 inch thick.

In some embodiments, the top layer 116 further includes a first opening 136 through which the substrate contacts a planar region of the wicking pad downstream of the reservoir (fig. 1A-2B). In certain embodiments, the top layer 116 further includes a second opening 138 through which the pump contacts a second plane of the wicking pad downstream of the first opening 136. In some embodiments (fig. 3A-3C), the top layer 116 partially covers the wicking pad, i.e., only the reservoir covers and adheres to the wicking pad. In certain embodiments, there is no top layer downstream of the reservoir, i.e. in the planar area on which the substrate and pump are applied. In some embodiments, the top layer 116 includes an opening downstream of the reservoir, and the opening is of sufficient size to accommodate the application of both the substrate 112 and the pump 124 to the wicking pad 102.

The pump 124 is located at or near the second end 106 of the wicking pad 102 and is in intimate contact with the wicking pad 102. The dry pump 124 serves as a drain by wicking fluid from the reservoir 122 through the wicking pad 102.

The planar region 110 of the wicking pad 102 may include graphics/indicia or other indications as to where the user should place the substrate 112 or where the adhesive is secured in/on the wicking pad. Alternatively, the graphics/indicia may be on a support layer of the device.

The wicking pad 102 has a width, a length, and a height (e.g., thickness). The wicking pad 102 may be of any size and shape. In certain embodiments, at least a portion of the wicking pad 102 (e.g., the planar area 110 for application of the substrate 112) is planar. In some cases, the length and width of the wicking pad 102 is at least about 2 times, 5 times, 10 times, 100 times, or more greater than the height (i.e., thickness).

Exemplary dimensions of the wicking pad include, but are not limited to, at least about 0.25cm, 0.5cm, 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 10cm, 12cm, 15cm, 20cm, 25cm, 30cm, or greater in at least one dimension. In some cases, the wicking pad 102 is 20± 0.5,1,2,3,4,5,6,9 or 10cm in length (in the direction of flow) and 10± 0.5,1,2,3,4,5,6,7,8 or 9cm in width.

The wicking pad 102 is an absorbent material. In some embodiments, the wicking pad 102 is configured to have a high solution capacity and lateral flow rate. In some cases, high solution capacity and lateral flow rates are provided by the wicking pad 102 having a substantial height (e.g., thickness). In some cases, the thickness of the wicking pad 102 is about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, or about 0.2mm. In some cases, the thickness of the wicking pad 102 is between about 0.05mm to about 0.5 mm.

In some embodiments, the wicking pad 102 also includes one or more regions separated from each other by a hydrophobic or impermeable barrier 542 that is parallel to the edges of the wicking pad (fig. 5). The barrier 542 inhibits, eliminates or substantially eliminates fluid communication (e.g., fluid flow) between adjacent regions in the wicking pad and allows for simultaneous processing of multiple substrates. Hydrophobic barriers include, but are not limited to, wax barriers, or barriers resulting from vapor or liquid phase silanization of the wicking pad. Exemplary materials that may form the impermeable barrier include, but are not limited to, waxes, plastics, polymers, and resins. In some embodiments, each zone has its own pump 124.

The wax used to form the wax barrier may be any wax that is flowable at elevated temperatures and non-flowable at room temperature (e.g., about 20-25 ℃). Examples are paraffin waxes, microcrystalline waxes, thermosetting waxes, animal waxes (such as beeswax, lanolin and tallow), vegetable waxes (such as soybean wax, carnauba wax, candelilla wax and palm wax), mineral waxes (such as ceresin and montan wax), petroleum waxes and synthetic waxes (such as olefinic polymers, chlorinated naphthalene and Fischer-Tropsch waxes). In addition to normal paraffins and isoparaffins, the paraffin composition may contain small amounts of naphthenes or olefins, or both. Useful waxes include waxes that become flowable (i.e., have a melting point) in a temperature range of about 60 ℃ to about 150 ℃, or about 75 ℃ to about 125 ℃. Wax formulations and compositions exhibiting this pattern are known to those skilled in the art.

The silylating agent used to form the hydrophobic barrier may be any silylating agent that reacts with the porous substrate or portion thereof. For example, if the porous substrate contains cellulose, a silylating agent that silylates the hydroxyl groups of the cellulose backbone may be used. Exemplary silylating agents include, but are not limited to, trimethylchlorosilane, trimethylsilane, or hexamethyldisilazane. The silylating agent also includes triethoxysilane (R- -Si (C) ₂ HSO) ₃ ) Wherein R is, for example, vinyl, methacryloyl, aminopropyl, fluoroalkyl or thioethyl. Other suitable silylating agents will be apparent to those skilled in the art. The polymer may react with silane groups to create an impermeable barrier.

A wax or other barrier-forming agent (e.g., a silylating agent or an impermeable barrier) may be applied to one or both sides of the porous substrate, although in most cases, the application to one side will be sufficient to allow the porous substrate to permeate, or to allow permeation (e.g., by melting after application), the porous substrate to some extent be sufficient to act as a barrier to liquid flow. The barrier-forming agent may be applied as a liquid. The liquid may be applied by hand or by other means. In some cases, the liquid is sprayed or poured onto the porous substrate. The spraying may be accomplished with an ink jet printer or similar device. In some cases, the liquid hardens after application to form an impermeable and/or hydrophobic barrier. Alternatively, the barrier-forming agent may be applied as a gas. For example, the silylating agent, wax, plastic, resin or polymer may be applied in vapor form that condenses on or reacts with the porous substrate. Alternatively, the barrier-forming agent may be applied in solid form. For example, the wax may be applied as a solid manually or in an automated or mechanical manner. In some cases, the porous substrate is masked to protect the regions from the barrier-forming agent, and the barrier-forming agent is in contact with the porous substrate.

The application of the wax can be effected by hand (using a conventional crayon or wax pen) or by means of a wax printer (wax printer). Wax pens are known in the art and generally include a housing having a reservoir containing hot wax, a nozzle, and a handle. The application of the hot wax is achieved by pouring the housing so that the liquefied wax passes through the spout, and the housing is equipped with a valve to prevent the wax from flowing at the end of the printing line.

Wax printers are also known in the art and typically operate by thermal transfer printing using a printhead that includes a very small array of heating elements that are independently activated under software control to locally heat the wax above its melting point to release the wax to the print medium. Examples of commercially available wax printers include Phaser 8560DN (Fuii Xerox, ltd.) and CALCOMP COLORMASTER PLUS thermal wax transfer printers (CalComp Graphics, LLC), foothill Ranch, calif., U.S. A.A.. Wax printers and their use are described in U.S. patent No. 5,957,593 (9/28/1999) to Kroon (Tektronix, inc.); U.S. patent No. 8,206,664 (6/26/2012) to Lin (schle corporation (Xerox Corporation)); lu, y., et al, "Rapid prototyping of paper-based microfluidics with wax for low-cost, portable bioassay (rapid prototyping of paper-based microfluidics using wax for low cost portable biological assays)" electrophorsis 2009, 30, 1497-1500; and Carrilho, e., et al, "Understanding Wax Printing: a Simple Micropatterning Process for Paper-Based Microfluidics (understanding wax printer: simple microimaging process for paper-based microfluidics) "anal. Chem.,2009, 81 (16), 7091-7095. The width of the wax line applied (prior to heating) can vary and is not important in the present invention provided that the wax content of the line is sufficient to penetrate the porous substrate and form a barrier to the lateral flow of fluid within the porous substrate.

In some embodiments, once applied, the wax can be passed through the thickness of the porous substrate by heating the wax above its melting point to fill the pores or voids and form a lateral barrier to aqueous fluid flow. In some cases, the amount of wax applied will be such that the molten wax fully penetrates the thickness of the wicking pad, while the side flow of molten wax (i.e., in a direction parallel to the planar face of the sheet) is minimal or at least limited to a small distance that is substantially uniform along the length of the applied wax line, such that the resulting area defined by the wax barrier is known and controlled. Forming the barrier in this manner may also be controlled by the degree of heating, including the temperature at which the wax is heated and the length of time that heating is continued. The optimum temperature and duration can be readily determined by routine experimentation and error, but in most cases useful results are obtained by heating to at least 5 ℃ above the melting point of the wax, and in many cases about 5 to about 50 ℃ above the melting point of the wax, or about 10 to about 30 ℃ above the melting point. The most appropriate heating time depends on the temperature, the higher the temperature the shorter the time required. Typically, a heating time of about 15 seconds to about 20 minutes (or in many cases about 30 seconds to about 10 minutes) can provide useful results. Heating may be accomplished by conventional methods including radiant heating, conductive heating, convective heating, pulsed heating and microwave heating. The effective results can be achieved with a heated plate or a device as simple as a conventional oven.

The optimal width of the hydrophobic or impermeable barrier can vary depending on the size of the region bordered by the barrier and the thickness of the porous substrate and can be readily determined by routine testing. In most cases, the width ranges from about 10 microns to about 5 millimeters, from about 30 microns to about 3 millimeters, from about 100 microns to about 1 millimeter, or from about 200 microns to about 5 millimeters, or 10 millimeters.

In some embodiments (e.g., diagnostic assay embodiments), the wicking pad 102 has one or more reagents (e.g., binding agents, such as antibodies) immobilized or embedded therein in one or more portions (e.g., in one or more portions downstream of the reservoir). The embedded reagent is typically embedded or incorporated and dried into the wicking pad such that the reagent remains immobilized during fluid flow or such that the reagent does not move until contacted with the aqueous fluid front under side flow and released upon a user-defined event. These portions may be printed reagent lines or dots.

The wicking pad 102 typically has a large surface area due to the presence of a plurality of holes or voids. The large surface area may increase the loading capacity of the wicking pad 102 for one or more reagents or one or more reagent-containing solutions. In some embodiments, the wicking pad 102 has a thickness of at least about 0.001m ² /g、0.02m ² /g、0.1m ² /g、0.5m ² /g、1m ² /g、10m ² /g or higher, measured by standard techniques.

In some embodiments, the wicking pad 102 may have a particular pore size, a particular average pore size, or a particular range of pore sizes. For example, the wicking pad 102 may comprise 0.1 μm pores, 0.2 μm pores, 0.45 μm pores, or 1, 2, 4, 5, 6, 7, 8, 10, 15, 20 μm pores, or greater than about 20 μm pores. For another example, the wicking pad 102 may contain pores having an average size of 0.1, 0.2, 0.45, 1, 2, 4, 5, 6, 7, 8, 10, 15, or 20 μm or more. As another example, the wicking pad 102 may contain pores ranging in size from about 0.1-8 μm, 0.2-8 μm, 0.45-8 μm, 1-8 μm, 0.1-4 μm, 0.1-2 μm, 0.1-1 μm, 0.1-0.45 μm, 0.2-8 μm, 0.2-4 μm, 0.2-2 μm, 0.2-1 μm, 0.2-0.45 μm, 0.45-8 μm, 0.45-4 μm, 0.45-2 μm, 0.45-1 μm. In some cases, the wicking pad 102 may contain pores less than about 20 μm. For example, the wicking pad 102 may comprise a material in which at least about 50%, 60%, 70%, 80%, 90% or more of the pores have a size of less than about 20, 15, 10 or 5 μm. In some cases, the pores in the wicking pad 102 are large enough to accommodate one or more proteins of average size (e.g., about 1 nm). For example, the size of the pores may be at least 1nm, at least 5nm, at least 10, 100 or 500nm. Alternatively, at least 50%, 60%, 70%, 80%, 90% or more of the pores may have a size greater than 1, 5, 10, 50, 100 or 500nm. As used herein, pore size may be measured in terms of radius or diameter. In some cases, the wicking pad 102 comprises a porous polyethylene, such as a porous polyethylene having a pore size of 0.2 to 20 microns, or 1 to 12 microns. The wicking pad 102 may have different pore sizes in different areas of the pad. For example, the wicking pad 102 may have a lateral flow region with different pore sizes or ranges of pore sizes. In some embodiments, the aperture is selected to control the flow rate. For example, a larger aperture would allow for a faster flow rate. In some cases, the wicking pad (e.g., fiberglass or cellulose) contains voids, which may be defined by the particle size and/or flow rate (e.g., the time it takes for water to flow 4 cm) of the material to remain.

In an embodiment, the edge of the wicking pad 102 has a water-tight seal 139 (see fig. 1a,2a,3a and 3B). The seal 139 is formed of a waterproof material or a heat seal to prevent leakage of liquid from the edge during lateral flow. Exemplary water resistant materials that can be used to seal the edges of the wicking pad include, but are not limited to, acrylics (e.g., nail polish), waxes, and photopolymers.

The wicking pad 102 may be treated or functionalized to minimize non-specific agent binding, increase lateral flow, increase wicking, or reduce protein aggregation. For example, the wicking pad 102 or portions thereof may be treated to alter the hydrophilicity or hydrophobicity of the treated region. In some cases, changing the hydrophilicity or hydrophobicity of the wicking pad 102 may increase the binder loading, reduce binder aggregation or denaturation, create masking zones (where the binder is excluded or not loaded) when the wicking pad is wet, or direct the binder flow. In some cases, the wicking pad contains a protein aggregation modifier as described herein.

The wicking pad 102 and pump are typically formed of a water absorbent material and may be made of, for example, natural fibers, synthetic fibers, fiberglass, or mixtures thereof. Non-limiting examples include nitrocellulose, cotton, glass, and combinations thereof. There are many commercially available materials for diagnostic use from suppliers including, but not limited to, alston (Ahlstrom), GE, PALL, miibos (Millipore), sartous (Sartorius), S & S.

The pump is formed of a material having a liquid absorbing capacity that is substantially greater than the wicking pad 102 and allows for a faster fluid flow rate than through the wicking pad 102. In some embodiments, the pump is formed from one or more absorbent pads (e.g., a water absorbent material).

The water absorbing material may include, but is not limited to, a polymer-containing material. The polymer may be in the form of polymer beads, polymer membranes or polymer monoliths. In some cases, the polymer is cellulose. The cellulose-containing pad comprises a paper, cloth, woven or nonwoven cellulose substrate. Cloth mats include those containing natural cellulosic fibers such as cotton or wool. Paper mats include those containing natural cellulosic fibers (e.g., cellulose or regenerated cellulose) and those containing cellulose fiber derivatives including, but not limited to: cellulose esters (e.g., nitrocellulose, cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate-butyrate, and cellulose sulfate), and cellulose ethers (e.g., methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylcellulose, hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose, ethylhydroxyethyl cellulose, and carboxymethyl cellulose). In some cases, the cellulosic pad contains rayon. In some cases, the pad is paper, e.g. of various kinds

Paper.

The water absorbing material may also include, but is not limited to, a sintered material. For example, the water absorbing material may comprise sintered glass, sintered polymer, or sintered metal, or a combination thereof. In some cases, the sintered material is formed by sintering one or more powdered glasses, powdered polymers, or powdered metals. In other cases, the sintered material is formed by sintering one or more glass, metal, or polymer fibers. In other cases, the sintered material is formed by sintering one or more glass, polymer, or metal beads.

The water-absorbing material may also contain, but is not limited to, one or more non-cellulosic polymers, such as synthetic polymers, natural polymers, or semi-synthetic polymers. For example, the material may contain polyesters, such as polyglycolide, polylactic acid, polycaprolactone, polyethylene adipateAlcohol esters, polyhydroxyalkanoates, polyhydroxybutyrate, poly (3-hydroxybutyrate-co-3-hydroxyvalerate), polyethylene terephthalate, polybutylene terephthalate, 1, 3-propanediol terephthalate, polyethylene naphthalate,

In some cases, the polymer is spunbond, such as spunbond polyester.

Other synthetic polymers include, but are not limited to: nylon, polypropylene, polyethylene, polystyrene, divinylbenzene, ethylene polymer (polyvinyl), polydifluoroethylene, high density polydifluoroethylene, polyacrylamide, (C) ₂ -C ₆ ) Mono-olefin polymer, vinyl aromatic polymer, vinyl amino aromatic polymer, vinyl halide polymer, (C) ₁ -C ₆ ) Alkyl (meth) acrylate polymer, (meth) acrylamide polymer, vinylpyrrolidone polymer, vinylpyridine polymer, (C) ₁ -C ₆ ) Hydroxyalkyl (meth) acrylate polymer, (meth) acrylic acid polymer, acrylamidomethylpropane sulfonic acid polymer, and N-hydroxyl group-containing (C) ₁ -C ₆ ) An alkyl (meth) acrylamide polymer, acrylonitrile, or a mixture of any of the foregoing.

The substrate 112 is generally planar in shape and may be a membrane formed, for example, of nitrocellulose, polyvinylidene fluoride, nylon, or polysulfone. Other materials from which the substrate 112 may be formed include, but are not limited to, glass, plastic, silicon, metal, and/or metal oxide, either bare or functionalized with a polymer. Plastic materials from which the substrate 112 may be formed include, but are not limited to, polyethylene terephthalate, polypropylene, polystyrene, and/or polycarbonate. Examples of polymers for functionalizing the surface of a substrate formed of plastic, exfoliated, metal or metal oxide include glycidoxypropyl triethoxysilane; poly-L-lysine; a polybrene; polyethylene glycol polymer; a dextran polymer; aminopropyl silane; carboxysilane (caroxicilane); hydrogels and polymer brushes; and/or self-assembled monolayers of, for example, functionalized alkyl thiols, dendrimers, or oligonucleotides.

Exemplary bonding methods for bonding all or part of the support layer and the top layer to the wicking pad include, but are not limited to, adhesive bonding, thermal bonding, and organic solvent bonding, with or without pressure. In embodiments where an adhesive is used, the nature of the adhesive may affect the performance of the test (i.e., flow characteristics, reagent stability, etc.) and may be optimized for the desired test or application. In some embodiments, the adhesive may be part of a support layer of the device 100. Exemplary adhesives include, but are not limited to, spray adhesives, ultraviolet light curable adhesives, or pressure sensitive adhesives.

In some embodiments, the support layer and/or the top layer are formed of a liquid impermeable material (e.g., plastic), including, but not limited to, polyethylene terephthalate, glycol modified polyethylene terephthalate, polypropylene, polystyrene, and polycarbonate. The support layer and/or the top layer may be, for example, vacuum or injection molded or otherwise constructed.

A. Exemplary detection reagents

i. Binding agent

Binding agents are described herein for detecting analytes. In some cases, the binding agent is an antibody (e.g., a primary or secondary antibody). An antibody may be used to bind an analyte. In some cases, the primary antibody is labeled such that the primary antibody, and thus the analyte, can be detected. In some cases, the primary antibody is detected by binding to a labeled secondary binding agent, such as a labeled secondary antibody. In some cases, tertiary binders are used to detect complexes containing analytes and primary binders as well as secondary binders.

The binding agent may be provided in one or more reagent solutions. The reagent solution may contain one or more buffers, salts, densitometers or protein aggregation modifiers as described herein. The density agent may be used to adjust the viscosity of the reagent solution, which will adjust the rate at which the solution flows out of the reservoir. Having a density agent in each reagent solution may also enhance the binding interaction between, for example, an analyte immobilized on a substrate and a binding agent (e.g., an antibody). Examples of densitometers include, but are not limited to, glycerol, sucrose, trehalose, dextran, and polyethylene glycol. The binding agent may be stored in solution for at least about 1 day, 3 days, 7-10 days, at least about 1 month, 2 months, 3 months, 6 months, 1 year, or longer.

The adhesive may also be provided on or in the wicking pad. For example, lines or dots of adhesive may be secured in/on the wicking pad downstream of the reservoir (e.g., in planar region 110). In some embodiments, the first binding agent is a reversibly immobilized labeled first antibody (e.g., a primary antibody conjugate) for detection, the second binding agent is an irreversibly immobilized unlabeled second antibody (e.g., a "test" primary antibody) for capture, and the third binding agent is a control antibody that binds to the first primary antibody. Control antibodies can be used to evaluate assay effectiveness. In certain embodiments, the labeled first antibody is paired with the second antibody, and the two antibodies bind different epitopes on the analyte such that the analyte (if present) is sandwiched between the first and second antibodies during the lateral flow assay. In some embodiments, a plurality of paired first and second primary antibodies are immobilized on a wicking pad to allow for multiplexed detection of analytes in a sample.

In some cases, the planar region of the wicking pad in fluid communication with the fluid in the one or more reservoirs comprises one or more binding agent dried thereon. The dried binder may be reconstituted by contacting the planar region of the wicking pad with an aqueous solution. In some cases, the aqueous reconstitution buffer may contain one or more rewetting reagents, including salts, buffers, or protein aggregation modifiers as described herein. In some cases, the binding agent may be stored dry or substantially dry in the wicking pad for at least about 1 day, 3 days, 7-10 days, at least about 1 month, 2 months, 3 months, 6 months, 1 year, or more.

ii. markers

Analytes can be detected by detecting labels attached to the binding agent. The label may be directly attached to the binding agent (e.g., by a covalent bond or other bond to the primary antibody) or may be indirectly attached (e.g., using a chelator or linker molecule). The terms "label" and "detectable label" are used synonymously herein. In some embodiments, each label (e.g., a first label attached to a first binding agent, a second label attached to a second binding agent, etc.) generates a detectable signal and these signals (e.g., a first signal generated by the first label, a second signal generated by the second label, etc.) are distinguishable. In some embodiments, the two or more binding agent labels comprise the same type of reagent (e.g., the first label is a first fluorescent agent and the second label is a second fluorescent agent). In some embodiments, two or more binding agent labels (e.g., a first label, a second label, etc.) combine to generate a detectable signal that cannot be generated in the absence of the one or more labels.

Examples of detectable labels include, but are not limited to: biotin/streptavidin labels, nucleic acid (e.g., oligonucleotide) labels, chemically reactive labels, fluorescent labels, enzyme labels, radioactive labels, quantum dots, polymer dots, mass labels, colloidal gold, electrochemical labels, and combinations thereof. In some embodiments, the label may include an optical agent, such as a chromophore, fluorescent agent, phosphorescent agent, chemiluminescent agent, or electrochemiluminescent agent. A variety of reagents (e.g., dyes, probes, or indicators) are known in the art and may be used in the present invention. (see, e.g., injetty (Invitrogen), the Handbook-A Guide to Fluorescent Probes and Labeling Technologies (Handbook-fluorescent probes and labeling techniques instructions, 10 th edition (2005)). Chromophores include coenzymes or cofactors having detectable absorbance. In some cases, the binding reagent can be detected by detecting the intrinsic absorbance of the peptide bond at, for example, 220nm or the absorbance of the complex amino acid at 280 nm.

The fluorescent agent may include various organic and/or inorganic small molecules or various fluorescent proteins and derivatives thereof. For example, fluorescent agents may include, but are not limited to: cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines, phenylxanthenes, phenothiazines, phenoselenzines, luciferins (e.g., FITC, 5-carboxyluciferin, and 6-carboxyluciferin), benzoporphyrins, squaraines, dipyrromethenes Pyridones, naphthacenes, quinolines, pyrazines, corrines, croconic acids, acridones, phenanthridines, rhodamines (e.g., TAMRA, TMR, and rhodamine red), acridines, anthraquinones, chalcogenium (chalcogenium) analogs, chlorins, naphthalocyanines, methine dyes, indolium dyes, azo compounds, chamomile blue, azachamomile blue, triphenylmethane dyes, indole, benzindole, indocarbocyanine, benzindole carbocyanine, BODIPY ^TM And BODIPY ^TM Derivatives, and analogs thereof. In some embodiments, the fluorescent agent is an Alexa Fluor dye, a DyLight dye, or an IRDye. In some embodiments, the fluorescent agent is a polymer dot or a quantum dot. Fluorescent dyes and fluorescent label reagents include those available from, for example, invitrogen/Molecular Probes (Invitrogen/Molecular Probes) (ewing, oregon), pierce biotechnology company (Pierce Biotechnology, inc.) (lokid, illinois), and Licor bioscience company (Licor Biosciences) (lincoln, inner boulder). In some embodiments, the optical agent is an intercalating dye. In some embodiments, 2, 3, 4, 5 or more binding agents are each labeled with an optical agent such as a fluorescent agent (e.g., a first binding agent is labeled with a first fluorescent label, a second binding agent is labeled with a second fluorescent label, etc.), and each binding agent labeled with an optical agent is detected by detecting a signal generated by the optical agent (e.g., a fluorescent signal generated by a fluorescent label). In some embodiments, the second fluorescent label quenches the fluorescent signal generated by the first fluorescent label. In some embodiments, the first and second fluorescent labels are members of a Fluorescence Resonance Energy Transfer (FRET) pair. The term "fluorescence resonance energy transfer" or "FRET" refers to energy transfer between at least two chromophores, a donor chromophore and an acceptor chromophore. Typically in FRET, if the donor and acceptor are sufficiently close, the donor transfers its energy to the acceptor when the donor is excited by optical radiation having a suitable wavelength. The receptor may re-emit the transferred energy in the form of optical radiation of a different wavelength. Suitable FRET pairs (donor/acceptor) include, but are not limited to EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein Cy 5, fluorescein/Cy 5.5, and fluorescein/LC Red705.

In some embodiments, all binding agents are labeled with an optical agent, and each binding agent labeled with an optical agent is detected by detecting a signal generated by the optical agent.

In some embodiments, the label is a radioisotope. Radioisotopes include radionuclides that emit gamma rays, positrons, beta and alpha particles, and X-rays. Suitable radionuclides include, but are not limited to: ²²⁵ Ac、 ⁷² As、 ²¹¹ At、 ¹¹ B、 ¹²⁸ Ba、 ²¹² Bi、 ⁷⁵ Br、 ⁷⁷ Br、 ¹⁴ C、 ¹⁰⁹ Cd、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁸ F、 ⁶⁷ Ga、 ⁶⁸ Ga、 ³ H、 ¹⁶⁶ Ho、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³⁰ I、 ¹³¹ I、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹³ N、 ¹⁵ O、 ³² P、 ³³ P、 ²¹² Pb、 ¹⁰³ Pd、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁴⁷ Sc、 ¹⁵³ Sm、 ⁸⁹ Sr、 ^99m Tc、 ⁸⁸ y and ⁹⁰ y. In some embodiments, 2, 3, 4, 5 or more binders are each labeled with a radioisotope (e.g., a first binder is labeled with a first radioisotope, a second binder is labeled with a second radioisotope, etc.), and each binder labeled with a radioisotope is measured by detecting the radioisotope-generated radioactivity. For example, one binder may be labeled with gamma emitters and one binder may be labeled with beta emitters. Alternatively, these binders may be labeled with radionuclides that emit the same particle (e.g., α, β, or γ) at different energies, where the different energies are distinguishable. In some embodiments, all of the binding agents are labeled with a radioisotope, and each labeled binding agent can be detected by detecting the radioactivity generated by the radioisotope.

In some embodiments, the label is an enzyme and the binding agent is detected by detecting a product produced by the enzyme. Examples of suitable enzymes include, but are not limited to: urease, alkaline phosphatase, (horseradish) Hydroperoxide (HRP), glucose oxidase, beta-galactosidase, luciferase, alkaline phosphatase, and esterases that hydrolyze luciferin diacetate. For example, the horseradish peroxidase detection system can be used in combination with a chromogenic substrate, tetramethylbenzidine (TMB), which produces a soluble product detectable at 450nm in the presence of hydrogen peroxide, or with a chemiluminescent substrate, such as Clarity from bioradiometric Laboratories (Bio-Rad Laboratories), which produces detectable light. The alkaline phosphatase detection system can be used in combination with a chromogenic substrate, p-nitrophenyl phosphate, which produces a soluble product that is readily detectable at 405 nm. The beta-galactosidase detection system can be used in combination with the chromogenic substrate o-nitrobenzene-beta-D-galactoside (ONPG), which produces a soluble product that is detectable at 410 nm. The urease detection system may be used in combination with a substrate such as urea bromocresol purple (sigma immunochemical company (Sigma Immunochemicals), st. In some cases, the enzyme acts on the fluorogenic substrate to produce a detectable fluorescent product. In some embodiments, 2, 3, 4, 5 or more binding agents are each labeled with an enzyme (e.g., a first binding agent labeled with a first enzyme, a second binding agent labeled with a second enzyme, etc.), and each binding agent labeled with an enzyme is measured by detecting a product produced by the enzyme. In some embodiments, all binding agents are labeled with an enzyme, and each binding agent labeled with an enzyme is detected by detecting the product produced by the enzyme.

In some embodiments, the label is an affinity tag. Examples of suitable affinity tags include, but are not limited to: biotin, peptide tags (e.g., FLAG tag, HA tag, his tag, myc tag, S tag, SBP tag, strep tag, eXact tag) and protein tags (e.g., GST tag, MBP tag, GFP tag).

In some embodiments, the label is a nucleic acid label. Examples of suitable nucleic acid markers include, but are not limited to: oligonucleotide sequences, single-stranded DNA, double-stranded DNA, RNA (e.g., mRNA or miRNA), DNA-RNA hybrids, or artificial nucleic acid analogs (e.g., LNA, PNA). In some embodiments, the nucleic acid marker is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length. In some cases, the nucleic acid label is an amplified nucleic acid (e.g., by PCR or by isothermal polymerase extension). In some cases, one or more labels are incorporated into the nucleic acid label using a polymerase, reverse transcriptase, ligase, or other enzyme that acts on the nucleic acid (e.g., fluorescence modified nucleotide, biotin-nucleotide, digoxigenin-nucleotide, hapten nucleotide). In some embodiments, a nucleic acid label is linked to another label (e.g., a nucleic acid) to produce a detectable product (e.g., a proximity ligation assay).

In some embodiments, the label is a nucleic acid barcode. As used herein, a "barcode" is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, or 12 nucleotides in length) that specifically defines a labeled molecule or a second molecule that binds to a labeled binding agent. The length of the barcode sequence determines how many unique samples can be distinguished. For example, a 4 nucleotide barcode can distinguish no more than 4 ⁴ I.e., 256 samples; a 6 nucleotide barcode can distinguish no more than 4096 different samples; whereas an 8 nucleotide barcode can index no more than 65,536 different samples. The application of barcode technology is well known in the art, see for example Katsuyuki Shiroguchi et al, "Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes (optimized single molecule barcodes for digital RNA sequencing minimizing sequence dependent bias and amplification noise)", PNAS (2012, 1 month, 24 days); 109 (4): 1347-52; and Smith, AM, et al, "Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples (highly multiplex barcode sequencing: a highly efficient method of parallel analysis of pooled samples) ", nucleic Acids Research (month 7 2010); 38 (13): e142.

In some embodiments, the label is a "click" chemistry moiety. Click chemistry uses simple robust reactions (such as copper-catalyzed cycloaddition of azides and alkynes) to establish intermolecular linkages. For a review of click chemistry, see Kolb et al, agnew Chem 40:2004-2021 (2001). In some embodiments, click chemistry moieties (e.g., azide or alkyne moieties) can be detected using another detectable label (e.g., a fluorescently labeled, biotinylated, or radiolabeled alkyne or azide moiety).

Techniques for linking a detectable label to a binding agent such as a protein (e.g., an antibody) are well known. For a review of common protein labelling techniques, see Biochemical Techniques: theory and Practice (biochemistry: theory and practice), john F. Robyt and Bernard J. White, wilfland Press, inc. (1987). Other labeling techniques are reviewed, for example, in r.haugland, excited States ofBiopolymers (excited state of biopolymers), steiner, planan Press (1983); fluorogenic Probe Design and Synthesis: a Technical Guide (fluorescent Probe design and Synthesis: technical guidelines), PE applied biosystems (PE Applied Biosystems) (1996); and G.T.Herman, bioconjugate Techniques (bioconjugate technology), academic Press (Academic Press) (1996).

In some embodiments, two or more labels (e.g., a first label, a second label, etc.) combine to generate a detectable signal that is not generated when one or more of the plurality of labels is absent. For example, in some embodiments, each label is an enzyme, and the activities of these enzymes combine to generate a detectable signal to indicate the presence of the label (and thus the presence of each labeled protein). Examples of enzymes that jointly generate a detectable signal include paired assays, such as paired assays using hexokinase and glucose-6-phosphate dehydrogenase; and chemiluminescent assays for NAD (P) H coupled to glucose-6-phosphate dehydrogenase, beta-D-galactosidase or alkaline phosphatase assays. See, e.g., maeda et al, J Biolumin Chemilumin 1989,4:140-148.

B. Protein aggregation modifier

Protein aggregation modifiers are described herein. Protein aggregation modifiers may be employed to reduce or eliminate aggregation or denaturation of binding agents such as proteins (e.g., antibodies) stored in or delivered from the reagent solution or wicking pad 102. For example, protein aggregation modifiers may be used to reduce or eliminate aggregation or denaturation of primary antibodies stored in or delivered from the reagent solution or wicking pad 102. In some cases, protein aggregation modifiers may be used to facilitate lateral flow of the binding agent in the lateral flow region 110 of the wicking pad 102 or to reduce background on the substrate 112.

In some cases, those protein aggregation modifiers that function to displace proteins out of the water-air interface and thereby protect the proteins from denaturation and aggregation are particularly effective in reducing aggregation of the binding agent immobilized on the wicking pad 102. In other cases, the protein aggregation modifier directly affects the stability of the binding agent by binding to and/or stabilizing the binding agent. In other cases, the protein aggregation modifier acts to shift the equilibrium away from the denatured or unfolded state and thereby reduce aggregation. For example, in some cases, interactions between the (disafanored) protein aggregation modifier and the binding agent are thermodynamically unfavorable due to strong repulsion between the amide backbone of the binding agent and the protein aggregation modifier. Thus, unfolding of the binding agent is not favored in the presence of the protein aggregation modifier, as unfolding exposes more of the amide backbone surface to the protein aggregation modifier.

The protein aggregation modifier may be one or more of the following: cyclodextrin, nonionic surfactant, ionic surfactant, zwitterionic surfactant, non-detergent sulfobetaines, simple sugars, polysaccharides, polyols, organic solvents, aggregation modified proteins, disordered peptide sequences, amino acids, redox agents, lyoprotectants, cryoprotectants, or chaotropes.

The cyclodextrin may be, but is not limited to: α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin, (2, 3, 6-tri-O-methyl) - β -cyclodextrin, (2-hydroxy) propyl- γ -cyclodextrin, random methyl- β -cyclodextrin, random methyl- γ -cyclodextrin, carboxymethyl- β -cyclodextrin, carboxymethyl- γ -cyclodextrin, 6-mono-deoxy-6-mono-amino- β -cyclodextrin, sulfobutyl- β -cyclodextrin, 6-amino-6-deoxy- β -cyclodextrin, acetyl β -cyclodextrin, succinyl α -cyclodextrin, succinyl β -cyclodextrin, succinyl γ -cyclodextrin, (2, 3, 6-tri-O-benzoyl) - β -cyclodextrin, succinyl- (2-hydroxypropyl) - β -cyclodextrin or succinyl- (2-hydroxypropyl) - γ -cyclodextrin. The cyclodextrin may also be a cyclodextrin polymer comprising one or more of the aforementioned cyclodextrin molecules. Other cyclodextrins are known in the art and include, for example, those described on the world wide web cyclindoxtrin. Exemplary concentrations of cyclodextrin are, but are not limited to, about 1mM, 2mM, 2.5mM, 5mM, 7.5mM, 10mM, 15mM, 20mM, 25mM, 50mM, 75mM, or 100mM.

The nonionic surfactant may be polyethylene-sorbitan-fatty acid ester, polyethylene-polypropylene glycol (polypropylene glycols) or polyoxyethylene-stearate. The polyethylene-sorbitan-fatty acid ester may be polyethylene (20) -sorbitan ester (Tween 20) ^TM ) Or polyoxyethylene (20) -sorbitan monooleate (Tween 80) ^TM ). The polyethylene glycol-polypropylene glycol may be a polyoxypropylene-polyoxyethylene block copolymer, for example, under the trade name

Or a Poloxamer ^TM Those sold. Polyoxyethylene-stearates may be, for example, under the trademark Myrj ^TM Those sold. Exemplary polyoxyethylene monolauryl ethers include those under the trade mark Brij ^TM Those sold, for example Brij-35. Exemplary concentrations of nonionic surfactants are, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5% or about 10% w/w, w/v or v/v.

The ionic surfactant may be an anionic surfactant or a cationic surfactant. Anionic surfactants useful in the present invention may be, but are not limited to, soaps, including alkali metal soaps, such as sodium, potassium or ammonium salts of aliphatic carboxylic acids (typically fatty acids), such as sodium stearate. Other anionic surfactants include organic amine soaps, such as organic amine salts of aliphatic carboxylic acids (typically fatty acids), such as triethanolamine stearate. Cationic surfactants useful in the present invention include, but are not limited to, ammonium salts such as octadecyl ammonium chloride or quaternary ammonium compounds such as benzalkonium chloride. The ionic surfactant may comprise sodium, potassium or ammonium salts of alkyl sulphates, such as sodium dodecyl sulphate or sodium octyl sulphate. Exemplary concentrations of ionic surfactants are, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5% or about 10% w/w, w/v or v/v.

Zwitterionic surfactants have cationic and anionic centers attached to the same molecule. For example, the cationic moiety is based on a primary, secondary or tertiary amine or a quaternary ammonium cation. The anionic moiety may be a sulfonate, as in CHAPS (3 [ (3-cholamidopropyl) dimethylammonium ] -1-propanesulfonate). Other anionic groups are sulfobetaines, such as cocamidopropyl hydroxysulfobetaine, or betaines, such as cocamidoethyl betaine, cocamidopropyl betaine or lauramidopropyl betaine. Exemplary concentrations of zwitterionic surfactants are, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5% and about 10% w/w, w/v or v/v.

Non-detergent sulfobetaines (NDSB) have short hydrophobic groups and sulfobetaine hydrophilic groups that cannot aggregate to form micelles, and thus NDSB is not considered a detergent. Exemplary NDSBs include, but are not limited to, NDSB 256, NDSB 221, NDSB 211, NDSB 201, NDSB 195, 3- (4-tert-butyl-1-pyrido) -1-propanesulfonate, 3- (benzyldimethylammonium) propanesulfonate, or dimethylethylammonium propanesulfonate. Exemplary concentrations of NDSB include, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5% and about 10% w/w, w/v or v/v.

Polyols are compounds having multiple hydroxyl functional groups. In some cases, polyols can alter the aggregation or denaturation behavior of proteins through a variety of mechanisms. For example, in some cases, polyols can shift the equilibrium to the folded state by giving a thermodynamically unfavorable interaction with the protein backbone. Alternatively, in some cases, the polyol may bind to and stabilize the folded state of the protein.

The polyol may be a simple sugar, for example: sucrose, mannitol, sorbitol, inositol, xylitol, erythritol, glucose, galactose, raffinose or trehalose. The polyol may also be a polysaccharide, such as dextran, starch, hydroxyethyl starch, or a polymer containing one or more of the simple sugars described herein. Glycerol, ethylene glycol, polyethylene glycol, pentaerythritol propoxylate and pentaerythritol propoxylate, and combinations thereof are also exemplary polyols.

The organic solvents may be, but are not limited to, those known to inhibit denaturation, unfolding, or aggregation of one or more proteins. A variety of suitable organic solvents are known in the art. For example, the organic solvent may include ethanol, butanol, propanol, phenol, dimethylformamide, 2-methyl-2, 4-pentanediol, 2, 3-butanediol, 1, 2-propanediol, 1, 6-hexanediol, or dimethylsulfoxide.

The aggregation-modifying protein may be a protein known in the art to inhibit denaturation, unfolding or aggregation of one or more proteins. Exemplary aggregation-modified proteins include, but are not limited to, albumin, chaperones, and heat shock proteins. Albumin is a protein that is water-soluble, moderately soluble in concentrated salt solutions, and subject to thermal denaturation. Exemplary albumins include serum albumin (e.g., bovine, equine, or human serum albumin) or ovalbumin (e.g., egg albumin). Other exemplary aggregation-modified proteins include casein, gelatin, ubiquitin, lysozyme, or advanced embryogenesis abundant (LEA) proteins. LEA proteins include LEA I, LEA II, LEA III, LEA IV, LEA V or atypical LEA proteins. LEA proteins are known in the art and are described, for example, in Goyal K.et al, biochemical Journal 288 (part 1), 151-57, (2005).

The protein aggregation modifier may also be an amino acid. In some cases, the amino acid may act as an oxidation-reduction to maintain an appropriate oxidation potential for the protein immobilized on the substrate 112. Suitable redox amino acids include cysteine and cystine. Other amino acids act to reduce denaturation or aggregation by non-redox methods. For example, arginine, glycine, proline and taurine have been shown to reduce protein aggregation.

Other redox agents may be used to reduce protein aggregation. Redox agents other than cysteine and cystine may be used to optimize the reduction potential in the substrate 112 to which the protein is immobilized. Exemplary redox agents include mercaptoethanol, dithiothreitol, dithioerythritol, tris (2-carboxyethyl) phosphine, glutathione disulfide and oxidized derivatives thereof, and Cu ²⁺ 。

The protein aggregation modifier may also include a lyoprotectant, a cryoprotectant, or a chaotrope. In some cases, the protein aggregation modifier is a chaotropic agent, such as urea, thiourea, a guanidine salt, a cyanate, thiocyanate, trimethylammonium, tetramethylammonium, cesium, rubidium, nitrate, acetate, iodide, bromide, trichloroacetate, or perchlorate. Under certain conditions, such as at low concentrations, chaotropic agents can reduce protein aggregation. Other protein aggregation modifiers include trimethylamine N-oxide.

The protein aggregation modifier may be a salt. Exemplary salts include, but are not limited to, sodium, potassium, magnesium, or calcium salts of hydrochloric, sulfuric, or phosphoric acid. The protein aggregation modifier may also be a buffer. Exemplary buffers include, but are not limited to TRIS (hydroxymethyl) aminomethane (TRIS), TAPSO, MES, HEPES, PIPES, CAPS, CAPSO, MOPS, MOPSO, or sodium or potassium phosphate, sodium or potassium carbonate, sodium or potassium bicarbonate, sodium or potassium citrate, sodium or potassium acetate, or sodium or potassium borate buffers.

The protein aggregation modifier may be provided in any suitable concentration. In some cases, the protein is provided in the form of an aqueous solution containing a binding agent and a protein aggregation modifier. In this case, the solution may be contacted with the wicking layer and optionally dried. Exemplary concentrations of protein aggregation modifier in aqueous binder solutions include, but are not limited to, about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 4%, 5%, 10%, 20% or about 25% or more w/v of the solution. Other exemplary concentrations include, but are not limited to, about 1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M, 75. Mu.M, 100. Mu.M, 150. Mu.M, 200. Mu.M, 300. Mu.M, 500. Mu.M, 750. Mu.M, 1mM, 5mM, 10mM, 25mM, 50mM, 100mM, 150mM, 200mM, 300mM, 500mM, and 1M.

In some cases, the protein aggregation modifier is provided in a reagent solution. Exemplary compositions containing protein aggregation modifiers contain about 0.001 wt%, 0.005 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt% or about 10 wt%, 20 wt% or about 25 wt% of one or more protein aggregation modifiers.

The protein aggregation modifier may be provided in any suitable combination. For example, in some cases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the foregoing protein aggregation modifiers may be used to reduce aggregation of the binding reagent immobilized on the wicking pad. In some cases, the wicking pad contains a protein aggregation modifier prior to contacting the wicking pad with the binder solution, and the binder solution contains the same or a different protein aggregation modifier. In some cases, the wicking pad contains a protein aggregation modifier prior to contacting the wicking pad with the binder solution, and the binder solution is free of protein aggregation modifier. In some cases, the binding reagent solution contains a protein aggregation modifier prior to contacting the wicking pad with the binding reagent solution, and the wicking pad or the area to be contacted is free of protein aggregation modifier.

Method

Methods of performing lateral flow assays using the devices described herein are provided. In one embodiment, the method includes contacting a substrate 112 (e.g., western blot) having immobilized analytes or binding agents in or on the wicking pad 102 with, for example, a lateral flow buffer, which substrate 112 may be provided pre-moistened or may be pre-moistened by a user. In some embodiments (fig. 1A-5), the substrate 112 is placed face down on the wicking pad 102 (e.g., between the reservoir and pump or in the planar region 110 of the wicking pad 102) through openings in the top layer 116 downstream of the reservoir and upstream of the pump 124.

Next, a different reagent solution is applied to each reservoir, starting from the first reservoir R1 closest to the region 110, for contacting the substrate 112. The reagent solutions may be applied to the reservoirs sequentially or simultaneously. The reagent solutions may also be applied to the reservoirs in any order. In one embodiment, four different reagent solutions (e.g., primary antibody, primary wash solution, secondary antibody or secondary detection reagent, and secondary wash solution) are applied to the reservoir. In embodiments having two or more sets of reservoirs (fig. 4 and 5), different or the same set of four reagent solutions are applied to each set of reservoirs depending on the analyte or binding agent immobilized on the substrate 112.

In some embodiments, a first reagent solution having a labeled primary antibody is applied to the first reservoir R1 and a second reagent solution having a first wash solution is applied to the second reservoir R2. In certain embodiments, four different reagent solutions are applied to the reservoir in the following order: a first reagent solution with a primary antibody is applied to the first reservoir R1, a second reagent solution with a first wash solution is applied to the second reservoir R2, a third reagent solution with a secondary antibody or secondary detection reagent is applied to the third reservoir R3, and a fourth reagent solution with a second wash solution is applied to the fourth reservoir R4. In some embodiments, the reagent solution applied to the reservoir has at least twice the volume of another reagent solution. For example, the volume of the second wash solution in the fourth reservoir R4 may be at least twice the volume of the second antibody in the third reservoir R3. In some embodiments, the fourth reagent solution with the second wash solution is omitted to allow more time for the secondary antibody or secondary detection reagent in the third reservoir R3 to bind the primary antibody.

In embodiments where the substrate has immobilized binding agents on it, the sample with analyte is applied to the first reservoir R1, the first wash solution is applied to the second reservoir R2, the secondary detection reagent is applied to the third reservoir R3 and, if desired, the second wash solution is applied to the fourth reservoir R4.

In some embodiments, in which there is no substrate 112 and in which the binding agent is immobilized on a line or spot on the planar region 110 of the wicking pad 102 downstream of the reservoirs, different solutions (e.g., sample or reagent solutions) are applied to at least two reservoirs. In one embodiment, in which a series of labeled, reversibly immobilized primary antibodies (e.g., primary anti-conjugates), a series of unlabeled, irreversibly immobilized secondary antibodies (e.g., test primary antibodies), and a series of irreversibly immobilized control antibodies that bind primary antibodies are printed onto the planar region 110 of the wicking pad 102, a sample with one or more analytes and optionally a control protein is applied to the first reservoir R1 and a wash solution (e.g., lateral flow buffer) is applied to the second reservoir R2. In some embodiments in which the unlabeled second anti-primary and control antibodies are irreversibly immobilized on the planar region 110 of the absorbent pad 102, a detection reagent (e.g., a labeled primary antibody) is applied to the third reservoir R3, and if desired, a second wash solution is applied to the fourth reservoir R4.

In embodiments where the analyte is immobilized in a line or spot on the planar region 110 of the wicking pad 102 downstream of the reservoirs, the labeled primary antibody is applied to the first reservoir R1 and the first wash solution is applied to the second reservoir R2. If necessary, a second detection reagent is applied to the third reservoir R3 and a second wash solution is applied to the fourth reservoir R4.

The reagent solution and/or sample is then flowed from the reservoir sequentially onto the planar region 110 of the wicking pad 102. In some cases, one or more dyes or indicators used in each reagent solution visually monitor the side flow (e.g., process) of each reagent solution out of reservoir 122 and into/through wicking pad 102.

In embodiments having an analyte immobilized on a substrate 112, the reagent solution is pushed by wicking from the reservoir into the wicking pad and dry pump, carrying the reagent (e.g., primary antibody, primary wash solution, and secondary antibody and second wash solution, if desired) in turn through a lateral flow into contact with the substrate 112 on which the protein or analyte is immobilized. The primary antibody in the first reagent solution is transported in the wicking pad 102, contacts the protein or analyte on the substrate 112, and binds to the target protein or analyte (if present) on the substrate 112. In some embodiments, the lateral flow of the reagent solution/lateral flow buffer from the reservoir to the pump also allows the first wash solution in the second reagent solution to be transported in the wicking pad 102 such that unbound primary antibody is removed from the substrate 112. In certain embodiments, the side flow of the reagent solution/side flow buffer from the reservoir to the pump also allows the secondary antibody or second detection reagent in the third reagent solution to be transported in the wicking pad 102 and contact the primary antibody that binds to the target protein (if present) on the substrate 112. In some embodiments, the lateral flow of the reagent solution/lateral flow buffer from the reservoir to the pump also allows the second wash solution in the fourth reagent solution to be transported in the wicking pad 102 such that unbound secondary antibodies are removed from the substrate 112. In some embodiments, the volume of the second wash solution applied to the wicking pad 102 and transported in the wicking pad 102 is twice the volume of the secondary antibody applied to the wicking pad 102 and transported in the wicking pad 102.

In embodiments in which the binding agent is immobilized on the substrate 112, the analyte (and optionally a control protein) is carried in the sample and reagent (e.g., first wash solution, secondary detection reagent and, if desired, second wash solution) by wicking from the reservoir into the wicking pad and dry pump to push the sample and reagent solution into sequential lateral flow contact with the substrate 112 in the reagent solution. In some embodiments, the sample is a biological sample. The biological sample may be obtained from any organism, such as an animal, plant, fungus, bacteria, or any other organism. In some embodiments, the biological sample is from an animal, such as a mammal (e.g., a human or non-human primate, cow, horse, pig, sheep, cat, dog, mouse, or rat), bird (e.g., chicken), or fish. The biological sample may be any tissue or body fluid obtained from an organism, for example blood, blood components or blood products (e.g. serum, plasma, platelets, red blood cells, etc.), sputum or saliva, tissue (e.g. kidney, lung, liver, heart, brain, nerve tissue, thyroid, eye, skeletal muscle, cartilage or bone tissue); cultured cells, e.g., primary cultures, explants, transformed cells, stem cells, feces or urine. In some embodiments, the sample includes a positive control protein for assessing assay effectiveness or for normalizing the test signal over a plurality of different antibody regions.

In embodiments in which the binding agent is immobilized on the region 110 of the wicking pad downstream of the reservoir, the sample and reagent (e.g., the first wash solution and, if desired, the secondary detection reagent and the second wash solution) carry the analyte by wicking from the reservoir to the wicking pad and dry pump to push the sample and reagent solution into sequential lateral flow contact with the region 110.

In some embodiments, a substantially uniform pressure is applied to the pump before or after beginning the lateral flow and during the lateral flow to improve the contact of the pump with the wicking pad 102. For example, a weight may be placed on top of the pump to advance the (urge) pump toward the wicking pad 102.

In some embodiments, a cap may be placed on the device once the reagent solution is applied to the reservoir to minimize evaporation and apply uniform pressure to the pump 124. The lid may be loosely placed on top of the device and then the capped device may be placed into a drawer-type container, which slides into the box. Prior to placing the cap on the device or cap location, a sponge may be placed on the pump to help apply uniform pressure to the pump. This process requires minimal user interaction with the consumable.

In some embodiments with a substrate, the binding of the primary antibody to the target protein (and optionally the contact of the secondary antibody or second detection reagent with the primary antibody) is tracked during lateral flow by visual inspection or using a detector. In some embodiments, the substrate is removed from the lateral flow device 100 and binding of the primary antibody to the target protein (if present) is detected. In some embodiments, antibodies that bind to a target protein are visualized and/or detected by using a detectable moiety and/or label as described herein. Suitable labels and/or moieties are detected by spectroscopic, photochemical, biochemical, immunochemical, isotopic, electrical, optical, chemical or mass spectrometry techniques.

In embodiments in which the binding agent is immobilized on the planar region 110 of the wicking pad, binding of the analyte (if present) to the first and second primary antibodies (e.g., detection of the analyte sandwiched between the first and second primary antibodies) during lateral flow is tracked by visual inspection or using a detector. In some embodiments, binding of the analyte to the first and second antibodies is visualized and/or detected by using a detectable moiety and/or label as described herein.

Many absorbent pad materials, wicking pad materials, and antibody-applying materials are known in the art, from which can be selected to control the volume, to control the flow rate of the system, to ensure uniform flow, and to include complete delivery of antibody/reagent from the reservoir. Other methods of influencing the timing of reagent/antibody delivery are possible, such as the use of a tortuous path in the wicking pad. In addition, other embodiments of controlling the lateral flow process may be engineered into the support layer, where the surface may contain sloped regions to slow or accelerate the flow of liquid using gravity.

Shown in fig. 1-3A is a consumable device that accommodates a single microgel sized membrane. Typically the user performs Western blots using what is known as a mid-blot, which is typically a 2x wide small membrane. In other Western blot applications, the user can cut the small and/or medium membranes into smaller portions, which correspond to several lanes of the original gel for protein electrophoresis and transfer (e.g., fig. 4). Thus, in some embodiments, the consumable lateral flow device may be sized to accommodate a small or medium size membrane. In other embodiments, a separate ridge may be molded into or otherwise present at the base of the consumable where the membrane portion may be placed. In embodiments with a hydrophobic or impermeable barrier in the wicking pad (fig. 5), the lateral flow region in the wicking pad may be sized to accommodate micro-and/or meso-scale membranes or membranes that have been cut into smaller portions.

In other embodiments of lateral flow devices, multiple antibodies may be mixed and loaded into one or more reservoirs to facilitate multiplexed detection of targets in a single sample.

IV. kit

Kits for performing lateral flow assays according to the methods described herein are provided. Kits comprising a lateral flow device as described herein are also provided. In some embodiments, the kit comprises reagents (e.g., binding agents including labeled primary or primary and secondary antibodies, wash solutions, and/or lateral flow buffers) in liquid form (e.g., reagent solutions) that are applied to the device by an end user. In some embodiments, the solution is provided in a concentrated form, e.g., 5x or 10x, which is diluted prior to use. In some embodiments, the reagents are provided in solid form, which is reconstituted with a liquid, e.g., a buffer, prior to use.

In some embodiments, the kit comprises a blocking agent (e.g., bovine serum albumin and/or skimmed milk powder), a surfactant (e.g., tween 20 or triton X-100), a protein aggregation modifier as described herein, a crowding agent (e.g., dextran, polyethylene glycol, and/or Ficoll), a density agent for controlling viscosity and fluid flow rate, and/or an agent that promotes uniform flow of the agent and/or promotes reaction to molecules on the substrate and minimizes background on the substrate. The additional reagents may be provided in the kit as a solid (e.g., powder) or in liquid form (e.g., as a solution). In some embodiments, the kit further comprises instructions for performing the methods described herein.

IV. examples

EXAMPLE 1 detection of ERK1/2 and beta-catenin from HeLa cell lysates

This example illustrates a lateral flow device as shown in fig. 1A-2B and as described herein for performing Western blot assays.

Thermal lamination (fig. 6A) or pressure sensitive adhesive (fig. 6B) was used to create the side-stream laminate used in the test. The heat sealing device was manufactured using a Scotch heat laminated pouch (3 m # tp3854-20), and in the second case a transparent polypropylene sheet protector and pressure sensitive adhesive (Lohmann GL 187) were used. An opening is cut into the top sheet of the pouch/protector to form an access point for introducing reagents (priAb, wash1, secAb, wash 2), the membrane to be probed and the dry pump/drain of the lateral flow device. For the reservoir, the slit was 1-4mm and 9cm long, the membrane was positioned at a 9cm x 8cm opening, and the pump was 4.5 x 9cm opening. By painting thin areas of a few millimeters with acrylic polymer (nail polish), the fiberglass wicking pad (10 cm x 26 cm) is sealed along the outer edges to close the voids of the fiberglass in these areas and prevent fluid from flowing out of the edges of the wicking pad. The sealed wicking pad is then inserted into the bag or pouch and aligned with the opening in the top surface. In the first case, scotch bags with aligned wicking pads are fed into a thermal lamination device to heat seal the top and bottom sheets of the bags around or over the wicking material; the heating also bonds the glass fibers to the bag. In the second case, pressure sensitive adhesives are applied to the top and bottom plastic sheet protective materials, and then these adhesives are applied to the top and bottom surfaces of the wicking material, encasing it. For the reservoir, rectangular through holes were cut into 3/8 inch acrylic sheets and the acrylic blocks were glued to the top plastic sheet.

For blotting, a stack of filter papers was placed in the pump position, the absorbent pad was wetted with Lateral Flow (LF) buffer (10 mM phosphate, 150mM NaCl,pH7.4,1% casein, 0.1% tween 20) by filling the reservoir and allowing the solution to wick onto the pump, and the membrane was then loaded with the protein side down into the membrane area. Rabbit primary antibody (cell signaling technologies Co. (Cell Signaling Technology)) was diluted 1:1000 in LF buffer and loaded (3 ml) into R1. 3ml of LF buffer was then loaded into R2, 3ml of secondary anti-goat anti-rabbit IgG-HRP (Bio-Rad STAR-208P) was loaded into R3 and 12ml of LF buffer was loaded into R4. After about 4 hours, the membrane was removed, washed in water for 2X 3 minutes and detected with Clarity chemiluminescent substrate (Bio-Rad). The blots were imaged using a ChemiDoc MP imager (burle corporation) with an exposure time of 60 seconds. The images in fig. 6A and 6B show a uniform and sensitive detection of analytes.

The results show that the lateral flow devices described herein can deliver Western blot reagents (e.g., specific binding agents, running buffers, wash solutions) in sequence and without user intervention on the blots of the wicking pads.

All patents, patent applications, and other published references cited in this specification are incorporated herein by reference in their entirety.

Claims (22)

1. A lateral flow device, comprising:

a wicking pad comprising a porous material, the wicking pad having a planar area for contacting a substrate comprising an immobilized analyte; and is also provided with

Wherein the wicking pad has a first end, a second end, and two side edges;

a support layer bonded to a first planar surface of the wicking pad;

a top layer bonded to the second planar surface of the wicking pad, the top layer comprising two or more reservoirs spatially separated from each other, wherein a longitudinal axis of each reservoir is perpendicular to a side edge of the wicking pad, wherein each reservoir is in fluid communication with the first end of the wicking pad, and wherein each reservoir comprises an aperture through which solution is released from the reservoir; and

a pump comprising an absorbent pad at a second end contacting the wicking pad,

wherein the top layer further comprises a first opening through which the substrate is in contact with a planar region of the wicking pad downstream of the reservoir, and wherein the top layer further comprises a second opening through which the pump is in contact with a second planar region of the wicking pad downstream of the first opening.

2. The device of claim 1, wherein the aperture is a slot.

3. The device of claim 1 or 2, wherein the reservoirs comprise two or more sets of reservoirs spatially separated from each other and adjacent to each other on a width axis of the lateral flow device.

4. The device of claim 3, wherein the wicking pad further comprises two or more regions separated from each other by a hydrophobic or impermeable barrier, the barrier being parallel to the side edges of the wicking pad.

5. The device of claim 1 or 2, wherein the top layer comprises an opening through which the substrate is in contact with the planar region and through which the pump is in contact with the wicking pad downstream of the planar region.

6. The device of claim 1 or 2, wherein the support layer and the top layer are formed of a liquid impermeable material.

7. The device of claim 6, wherein the liquid impermeable material is selected from the group consisting of polypropylene, polycarbonate, polystyrene, polyethylene terephthalate, and glycol-modified polyethylene terephthalate.

8. The device of claim 1 or 2, wherein the edges of the wicking pad are sealed with a waterproof material or heat seal.

9. The device of claim 8, wherein the water-resistant material is selected from the group consisting of acrylic, wax, and photopolymer.

10. The device of claim 1 or 2, wherein each reservoir spans the width of the wicking pad.

11. The device of claim 1 or 2, wherein at least a first reservoir shares a wall with at least a second reservoir.

12. The device of claim 1 or 2, wherein the analyte is a protein.

13. A kit for lateral flow comprising the device of any one of claims 1-12.

14. A method of performing a lateral flow assay, the method comprising:

providing an apparatus according to any one of claims 1-12;

applying a lateral flow buffer to the wicking pad;

applying a substrate comprising a protein to a planar region of the wicking pad;

starting from the reservoir closest to the planar area, applying a different reagent solution to each reservoir to apply the substrate; and is also provided with

The reagent solution is caused to flow from the reservoir side to the pump such that each reagent in the reagent solution is transported in turn in the wicking pad and contacts the proteins on the substrate.

15. The method of claim 14, wherein the step of allowing the lateral flow to occur comprises allowing the primary antibody from the first reagent solution in the first reservoir to bind to target protein if present on the substrate, and then allowing the first wash solution from the second reagent solution in the second reservoir to remove unbound primary antibody from the substrate.

16. The method of claim 15, wherein the step of allowing the lateral flow to occur further comprises contacting a secondary antibody or second detection reagent from a third reagent solution in a third reservoir with a primary antibody that binds to a target protein if present on the substrate.

17. The method of claim 16, wherein the step of allowing the lateral flow to occur further comprises allowing a second wash solution from a fourth reagent solution in a fourth reservoir to remove unbound secondary antibodies from the substrate.

18. The method of claim 17, wherein the volume of the second wash solution is at least twice the volume of the third reagent solution having the secondary antibody.

19. The method of any of claims 14-18, further comprising applying a substantially uniform pressure to the pump.

20. The method of any one of claims 15-18, further comprising removing the substrate after the primary antibody binds to the target protein if present and after the secondary antibody or secondary detection reagent is contacted with the primary antibody, and detecting binding of the primary antibody to the target protein if present.

21. The method of any one of claims 14-18, wherein different reagent solutions are applied to the reservoir sequentially.

22. The method of any one of claims 14-18, wherein different reagent solutions are applied to the reservoir simultaneously.

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